Eukaryotes chromosomes are subdivided into functionally and structurally autonomous domains. This was first recognized more than a half century ago in cytological studies on the lampbrush chromosomes of amphibian oocytes and the polytene chromosomes of insects. More recently, convincing evidence for the domain organization of eukaryotic chromosomes has come from a combination of genetic, molecular and biochemical experiments. These studies demonstrate that this organizing principle is of critical importance not only for the packaging of chromosomes inside the nucleus, but also for the proper regulation of gene activity. The subdivision of eukaryotic chromosome into domains requires a mechanism to separate one domain from another. Special elements called boundaries or insulators are thought to serve this purpose. Elements that function, as boundaries of chromatin domains were first identified in Drosophila, and they have now been found in a diverse array of organisms including yeast, sea urchins, Xenopus, chickens, mouse and humans. These elements define the limits of chromosomal domains and function to establish independent units of gene activity, insulating genes or regulatory elements within a domain from the action of regulatory elements located outside in adjacent domains. The aim of this proposal is to characterize several Drosophila boundaries and elucidate their mechanism of action.